Bottom Line:
This role depends on its ability to synthesize telomere repeats in a manner dependent on the reverse transcriptase (RT) function of its protein component telomerase RT (TERT), as well as on a novel pathway whose mechanism is poorly understood.Statistical comparisons to other microarray gene sets using pattern-matching algorithms revealed that the TERT transcriptional response strongly resembles those mediated by Myc and Wnt, two proteins intimately associated with stem cell function and cancer.These data show that TERT controls tissue progenitor cells via transcriptional regulation of a developmental program converging on the Myc and Wnt pathways.

Affiliation: Department of Medicine, Stanford School of Medicine, Stanford, California, United States of America.

ABSTRACTTelomerase serves a critical role in stem cell function and tissue homeostasis. This role depends on its ability to synthesize telomere repeats in a manner dependent on the reverse transcriptase (RT) function of its protein component telomerase RT (TERT), as well as on a novel pathway whose mechanism is poorly understood. Here, we use a TERT mutant lacking RT function (TERT(ci)) to study the mechanism of TERT action in mammalian skin, an ideal tissue for studying progenitor cell biology. We show that TERT(ci) retains the full activities of wild-type TERT in enhancing keratinocyte proliferation in skin and in activating resting hair follicle stem cells, which triggers initiation of a new hair follicle growth phase and promotes hair synthesis. To understand the nature of this RT-independent function for TERT, we studied the genome-wide transcriptional response to acute changes in TERT levels in mouse skin. We find that TERT facilitates activation of progenitor cells in the skin and hair follicle by triggering a rapid change in gene expression that significantly overlaps the program controlling natural hair follicle cycling in wild-type mice. Statistical comparisons to other microarray gene sets using pattern-matching algorithms revealed that the TERT transcriptional response strongly resembles those mediated by Myc and Wnt, two proteins intimately associated with stem cell function and cancer. These data show that TERT controls tissue progenitor cells via transcriptional regulation of a developmental program converging on the Myc and Wnt pathways.

pgen-0040010-g004: Acute Withdrawal of TERT Alters Expression of Genes Involved in Development, Signal Transduction, and Cell-to-Cell Signaling(A) In two cohorts of iK5-TERT mice used for microarrays, TERT was switched on postnatally, inducing anagen at day 60. In TERT ON, or control samples, iK5-TERT mice remained off doxycycline, and TERT expression was maintained (green arrow). In TERT OFF samples, iK5-TERT mice were injected with doxycycline at t = 0, acutely silencing TERT expression (red arrow).(B) Northern blots show rapid silencing of TERT mRNA after doxycycline injection in iK5-TERT mice. GAPDH serves as a loading control.(C) Clustered heat map of TERT-regulated gene expression profile (FDR < 0.05). Control = TERT ON; +Doxy = TERT OFF(D–E) Venn diagrams depicting the distribution of TERT-activated genes (D) or TERT-repressed genes (E) within each functional category.(F) List of TERT-activated genes analyzed in (D). Red, components of Bmp or Wnt pathways; green, cell-cycle–related genes; blue, genes having mouse epidermal phenotypes. Note that not all genes with known functions are color-coded.(G) List of TERT-repressed genes analyzed in (E).

Mentions:
Based on these data showing that TERT activates bulge stem cells, induces anagen, and stimulates proliferation in the IFE independent of catalytic function, we hypothesized that TERT acts in this context as a developmental regulator. We reasoned that we could identify putative pathways through which TERT acts by performing gene expression array experiments coupled with rigorous bioinformatic analyses. In designing these experiments, we leveraged several strengths of our system: (1) the ability to study the effects of TERT on the whole organ in vivo (2) temporal control of TERT with doxycycline enabling a study of dynamic gene expression changes over time and (3) a tetracycline-off configuration shown to result in rapid silencing in vivo [30]. To avoid the dramatic tissue changes associated with activating TERT and inducing anagen, we instead used iK5-TERT mice in which we allowed TERT to first induce anagen and hyper-proliferation of IFE. After induction of these changes, we then administered doxycycline to silence TERT expression, followed by a series of closely spaced serial biopsies. Because we were only interested in gene expression changes directly linked to TERT, we studied two cohorts of iK5-TERT mice, whose hair follicles were in TERT-induced anagen at age 60–65 days. In the TERT-off cohort, biopsies were obtained immediately before doxycycline treatment (t = 0), as well as 6, 12, and 24 hours after doxycycline injection to extinguish TERT expression. In parallel, age-matched iK5-TERT mice in the TERT-on cohort were injected with vehicle and biopsied at the same time points (Figure 4A). RNA was extracted from dorsal skin biopsies and used for Northern blot and for gene expression analyses using Affymetrix 430 2.0 arrays. TERT expression by Northern blot in the TERT-off cohort was efficiently suppressed by doxycycline injection within 6 hours (Figure 4B). This experimental design allows us to study the genome-wide response to acute withdrawal of TERT, while minimizing secondary gene expression changes caused by alterations in tissue architecture.

pgen-0040010-g004: Acute Withdrawal of TERT Alters Expression of Genes Involved in Development, Signal Transduction, and Cell-to-Cell Signaling(A) In two cohorts of iK5-TERT mice used for microarrays, TERT was switched on postnatally, inducing anagen at day 60. In TERT ON, or control samples, iK5-TERT mice remained off doxycycline, and TERT expression was maintained (green arrow). In TERT OFF samples, iK5-TERT mice were injected with doxycycline at t = 0, acutely silencing TERT expression (red arrow).(B) Northern blots show rapid silencing of TERT mRNA after doxycycline injection in iK5-TERT mice. GAPDH serves as a loading control.(C) Clustered heat map of TERT-regulated gene expression profile (FDR < 0.05). Control = TERT ON; +Doxy = TERT OFF(D–E) Venn diagrams depicting the distribution of TERT-activated genes (D) or TERT-repressed genes (E) within each functional category.(F) List of TERT-activated genes analyzed in (D). Red, components of Bmp or Wnt pathways; green, cell-cycle–related genes; blue, genes having mouse epidermal phenotypes. Note that not all genes with known functions are color-coded.(G) List of TERT-repressed genes analyzed in (E).

Mentions:
Based on these data showing that TERT activates bulge stem cells, induces anagen, and stimulates proliferation in the IFE independent of catalytic function, we hypothesized that TERT acts in this context as a developmental regulator. We reasoned that we could identify putative pathways through which TERT acts by performing gene expression array experiments coupled with rigorous bioinformatic analyses. In designing these experiments, we leveraged several strengths of our system: (1) the ability to study the effects of TERT on the whole organ in vivo (2) temporal control of TERT with doxycycline enabling a study of dynamic gene expression changes over time and (3) a tetracycline-off configuration shown to result in rapid silencing in vivo [30]. To avoid the dramatic tissue changes associated with activating TERT and inducing anagen, we instead used iK5-TERT mice in which we allowed TERT to first induce anagen and hyper-proliferation of IFE. After induction of these changes, we then administered doxycycline to silence TERT expression, followed by a series of closely spaced serial biopsies. Because we were only interested in gene expression changes directly linked to TERT, we studied two cohorts of iK5-TERT mice, whose hair follicles were in TERT-induced anagen at age 60–65 days. In the TERT-off cohort, biopsies were obtained immediately before doxycycline treatment (t = 0), as well as 6, 12, and 24 hours after doxycycline injection to extinguish TERT expression. In parallel, age-matched iK5-TERT mice in the TERT-on cohort were injected with vehicle and biopsied at the same time points (Figure 4A). RNA was extracted from dorsal skin biopsies and used for Northern blot and for gene expression analyses using Affymetrix 430 2.0 arrays. TERT expression by Northern blot in the TERT-off cohort was efficiently suppressed by doxycycline injection within 6 hours (Figure 4B). This experimental design allows us to study the genome-wide response to acute withdrawal of TERT, while minimizing secondary gene expression changes caused by alterations in tissue architecture.

Bottom Line:
This role depends on its ability to synthesize telomere repeats in a manner dependent on the reverse transcriptase (RT) function of its protein component telomerase RT (TERT), as well as on a novel pathway whose mechanism is poorly understood.Statistical comparisons to other microarray gene sets using pattern-matching algorithms revealed that the TERT transcriptional response strongly resembles those mediated by Myc and Wnt, two proteins intimately associated with stem cell function and cancer.These data show that TERT controls tissue progenitor cells via transcriptional regulation of a developmental program converging on the Myc and Wnt pathways.

Affiliation:
Department of Medicine, Stanford School of Medicine, Stanford, California, United States of America.

ABSTRACTTelomerase serves a critical role in stem cell function and tissue homeostasis. This role depends on its ability to synthesize telomere repeats in a manner dependent on the reverse transcriptase (RT) function of its protein component telomerase RT (TERT), as well as on a novel pathway whose mechanism is poorly understood. Here, we use a TERT mutant lacking RT function (TERT(ci)) to study the mechanism of TERT action in mammalian skin, an ideal tissue for studying progenitor cell biology. We show that TERT(ci) retains the full activities of wild-type TERT in enhancing keratinocyte proliferation in skin and in activating resting hair follicle stem cells, which triggers initiation of a new hair follicle growth phase and promotes hair synthesis. To understand the nature of this RT-independent function for TERT, we studied the genome-wide transcriptional response to acute changes in TERT levels in mouse skin. We find that TERT facilitates activation of progenitor cells in the skin and hair follicle by triggering a rapid change in gene expression that significantly overlaps the program controlling natural hair follicle cycling in wild-type mice. Statistical comparisons to other microarray gene sets using pattern-matching algorithms revealed that the TERT transcriptional response strongly resembles those mediated by Myc and Wnt, two proteins intimately associated with stem cell function and cancer. These data show that TERT controls tissue progenitor cells via transcriptional regulation of a developmental program converging on the Myc and Wnt pathways.